Steady-state acceptor fluorescence anisotropy imaging under evanescent excitation for visualisation of FRET at the plasma membrane

Devauges, Vivian, Matthews, Daniel R., Aluko, Justin, Nedbal, Jakub, Levitt, James A., Poland, Simon P., Coban, Oana, Weitsman, Gregory, Monypenny, James, Ng, Tony and Ameer-Beg, Simon M. (2014) Steady-state acceptor fluorescence anisotropy imaging under evanescent excitation for visualisation of FRET at the plasma membrane. PLoS One, 9 10: e110695-e110695. doi:10.1371/journal.pone.0110695


Author Devauges, Vivian
Matthews, Daniel R.
Aluko, Justin
Nedbal, Jakub
Levitt, James A.
Poland, Simon P.
Coban, Oana
Weitsman, Gregory
Monypenny, James
Ng, Tony
Ameer-Beg, Simon M.
Title Steady-state acceptor fluorescence anisotropy imaging under evanescent excitation for visualisation of FRET at the plasma membrane
Journal name PLoS One   Check publisher's open access policy
ISSN 1932-6203
Publication date 2014-10-31
Year available 2014
Sub-type Article (original research)
DOI 10.1371/journal.pone.0110695
Open Access Status DOI
Volume 9
Issue 10
Start page e110695
End page e110695
Total pages 16
Place of publication San Francisco, CA United States
Publisher Public Library of Science
Language eng
Formatted abstract
We present a novel imaging system combining total internal reflection fluorescence (TIRF) microscopy with measurement of steady-state acceptor fluorescence anisotropy in order to perform live cell Förster Resonance Energy Transfer (FRET) imaging at the plasma membrane. We compare directly the imaging performance of fluorescence anisotropy resolved TIRF with epifluorescence illumination. The use of high numerical aperture objective for TIRF required correction for induced depolarization factors. This arrangement enabled visualisation of conformational changes of a Raichu-Cdc42 FRET biosensor by measurement of intramolecular FRET between eGFP and mRFP1. Higher activity of the probe was found at the cell plasma membrane compared to intracellularly. Imaging fluorescence anisotropy in TIRF allowed clear differentiation of the Raichu-Cdc42 biosensor from negative control mutants. Finally, inhibition of Cdc42 was imaged dynamically in live cells, where we show temporal changes of the activity of the Raichu-Cdc42 biosensor.
Q-Index Code C1
Q-Index Status Provisional Code
Institutional Status Non-UQ

Document type: Journal Article
Sub-type: Article (original research)
Collections: Non HERDC
Queensland Brain Institute Publications
 
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